In mammalian cells, the MAPK (Mitogen-Activated Protein Kinase) signaling system is comprised of, at least, four distinct signaling modules defined by a core of MAP4K, MAP3K, MAP2K and MAPKs that are named after the “terminal” MAPK kinase in each pathway: ERK1/2, JNK1/2/3, p38alpha/beta, and ERK5 (Chang et al., 2001; Johnson et al., 2002; Pearson et al., 2001; and Raman et al., 2007). JNKs (c-Jun NH2-terminal kinase) become highly activated after cells are exposed to stress conditions such as cytokines, osmotic stress, hypoxia, and UV light, and are poorly activated by exposure to growth factors or mitogens (Derijard et al., 1994; and Pulverer et al., 1991). There are three distinct genes Jnk1, Jnk2, and Jnk3 that are alternatively spliced to yield approximately ten different proteins with the predominant isoforms: JNK1 and JNK2 expressed ubiquitously, and JNK3 expressed primarily in the nervous system (Derijard et al., 1994; Kallunki et al., 1994; Sluss et al., 1994; and Mohit et al., 1995). JNKs are activated by phosphorylation at the activation T-loop residues Thr183/Tyr185 by the MAP2Ks: MKK4 and MKK7, and are deactivated by MAP kinase phosphatases including MKP1 and MKP5. Signaling through the JNK-pathway is organized through binding to “scaffolding” proteins such as JIP which assemble signaling complexes containing MAP3K, MAP2K, and MAPKs in addition to transcription factors such as c-Jun, ATF2, and Elk1 which are phosphorylated by JNK. As JNKs comprise a central node in the inflammatory signaling network, it is not surprising that hyperactivation of JNK signaling is a very common finding in a number of disease states including cancer, inflammatory, and neurodegenerative diseases. A significant body of genetic and pharmacological evidence has been generated that suggest that inhibitors of JNK signaling may provide a promising therapeutic strategy. JNK3 knockout mice exhibit amelioration of neurodegeneration in animal models of Parkinson's and Alzheimer's disease (Kyriakis et al., 2001; Zhang et al., 2005; and Hunot et al., 2004). JNK1 phosphorylates IRS-1, a key molecule in the insulin-sensing pathway which down-regulates insulin signaling, and JNK1 knockout mice are resistant to diet-induced obesity (Aguirre et al., 2000 and 2002; Hirosumi et al., 2002; and Sabio et al., 2010). JNK2, often in concert with JNK1, has been implicated in the pathology of autoimmune disorders such as rheumatoid arthritis (Han et al., 2002) and asthma (Wong, W. S., 2005; Pelaia et al., 2005; Blease et al., 2003; Chialda et al., 2005); A recent study suggests that JNK2 may play a role in vascular disease and atherosclerosis as well (Osto et al., 2008). Yet, to date, no direct JNK inhibitors have been approved for use in humans.
Numerous small molecules from a variety of scaffolds such as indazoles, aminopyrazoles, aminopyridines, pyridine carboxamides, benzothien-2-ylamides and benzothiazol-2-yl acetonitriles, quinoline derivatives, and aminopyrimidines have been reported to act as selective ATP-competitive JNK inhibitors (LoGrasso and Kamenecka, 2008). However, despite this apparent plethora of reported JNK inhibitors, many exhibit poor kinase selectivity and/or do not inhibit the phosphorylation of well characterized substrates of JNK in cells. For example, one of the earliest and still most widely utilized inhibitors is the anthrapyrazolone, SP-600125 (Bennett et al., 2001) (FIG. 1) which exhibits exceptionally low specificity for JNK (Bain et al., 2007) and should only be used in combination with other approaches such as gene deletions or siRNA mediated depletion to rule-out a JNK role in a particular process (Inesta-Vaquera et al., 2010). Other reported JNK inhibitors such as AS601245 (Gaillard et al., 2005) only inhibit c-Jun phosphorylation at high concentrations which is likely due to a combination of limited cell penetration, ATP concentration, and differences between biochemical and cellular sensitivities to JNK inhibitors.